NIH Public Access Author Manuscript Biochem Pharmacol. Author manuscript; available in PMC 2009 January 1.

Transcription

1 NIH Public Access Author Manuscript Published in final edited form as: Biochem Pharmacol January 1; 75(1): Glutamatergic substrates of drug addiction and alcoholism 1 Justin T. Gass and M. Foster Olive Center for Drug and Alcohol Programs, Department of Psychiatry and Behavioral Sciences, Medical University of South Carolina, Charleston, SC USA Abstract The past two decades have witnessed a dramatic accumulation of evidence indicating that the excitatory amino acid glutamate plays an important role in drug addiction and alcoholism. The purpose of this review is to summarize findings on glutamatergic substrates of addiction, surveying data from both human and animal studies. The effects of various drugs of abuse on glutamatergic neurotransmission are discussed, as are the effects of pharmacological or genetic manipulation of various components of glutamate transmission on drug reinforcement, conditioned reward, extinction, and relapse-like behavior. In addition, glutamatergic agents that are currently in use or are undergoing testing in clinical trials for the treatment of addiction are discussed, including acamprosate, N-acetylcysteine, modafinil, topiramate, lamotrigine, gabapentin and mematine. All drugs of abuse appear to modulate glutamatergic transmission, albeit by different mechanisms, and this modulation of glutamate transmission is believed to result in long-lasting neuroplastic changes in the brain that may contribute to the perseveration of drug-seeking behavior and drug-associated memories. In general, attenuation of glutamatergic transmission reduces drug reward, reinforcement, and relapse-like behavior. On the other hand, potentiation of glutamatergic transmission appears to facilitate the extinction of drug-seeking behavior. However, attempts at identifying genetic polymorphisms in components of glutamate transmission in humans have yielded only a limited number of candidate genes that may serve as risk factors for the development of addiction. Nonetheless, manipulation of glutamatergic neurotransmission appears to be a promising avenue of research in developing improved therapeutic agents for the treatment of drug addiction and alcoholism. 1. Introduction Drug addiction is defined by several diagnostic criteria set forth by the American Psychiatric Association [1]. These criteria include a loss of control over drug intake, repeated unsuccessful attempts at quitting or reducing drug use, continued drug use despite negative consequences, a reduction in engagement in social, occupational and recreational activities in lieu of drugseeking or self-administration behavior, and the emergence of symptoms of tolerance or withdrawal. Historically, research into the neurobiological substrates that underlie the rewarding and reinforcing effects of drugs of abuse has focused on the mesolimbic dopamine reward circuitry, comprised primarily of dopaminergic neurons in the ventral tegmental area (VTA) that project rostrally to forebrain and limbic regions such as the nucleus accumbens 1 This work was supported by PHS grant AA (MFO) and AA (JTG) from the National Institute on Alcohol Abuse and Alcoholism, and a grant from the Alcoholic Beverage Medical Research Foundation (MFO). Corresponding author:, M. Foster Olive, Ph.D., Center for Drug and Alcohol Programs, Department of Psychiatry and Behavioral Sciences, 67 President Street, PO Box , Charleston, SC USA, Phone: , Fax: , olive@musc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

2 Gass and Foster Olive Page 2 (NAcc), amygdala and frontal cortex [2]. However, as can be seen in Fig. 1, there has been a dramatic increase in attention that has been given to the role of the excitatory amino acid glutamate in drug addiction and alcoholism over the past two decades. The purpose of this review is to summarize the effects of drugs of abuse on glutamatergic neurotransmission as well as key findings on the role of glutamate transmission in drug reinforcement, the rewarding effects of drugs of abuse, extinction of drug-seeking behavior, and relapse. Various glutamatergic medications that are either approved for clinical use or are being examined in clinical trials for the treatment of addictive disorders will also be discussed. Finally, a brief summary of findings on potential genetic linkages between individual components of glutamate neurotransmission and addiction is presented. 2. Animal models of drug addiction and alcoholism One of the most widely used methods to study drug addiction in animals is the intravenous self-administration (IVSA) paradigm [3]. In this model, experimental animals are implanted with indwelling intravenous catheters (most often the jugular vein in rodent studies) and are trained to perform an operant task (i.e., lever press or nose-poke) in order to receive an intravenous infusion of a cocaine, amphetamine, nicotine, etc. In the case of alcohol (ethanol), execution of the operant task results in presentation of a small amount of an alcohol-containing solution in a receptacle where the animal can consume the solution orally (some studies measure alcohol consumption in the home cage by presentation of two or more bottles containing ethanol solutions). By definition, if the delivery or presentation of the drug solution increases this behavior (i.e., appropriate lever pressing or nose-poking), the drug or alcohol solution is considered to be a positive reinforcer. Environmental cues, such as presentation of stimulus lights or auditory tones, are often paired with drug delivery or alcohol presentation to promote stimulus-drug associations, which enhance drug-taking behavior and can be utilized in subsequent reinstatement tests (see below). The effects of experimental manipulations (i.e., administration of test compounds either systemically or intracerebrally) on drug or alcohol self-administration behavior can then be assessed. However, the effects of any such manipulation must be interpreted with caution. For example, while it is tempting to interpret an observed decrease in self-administration behavior as signifying a reduction in the desire to self-administer the drug (and thus having possible therapeutic applications), there are equally plausible alternative explanations for the observed reduction in drug self-administration. For example, the experimental manipulation might have caused an overall reduction motor output, or an increase in sensitivity to the drug, resulting in less drug required to produce the same subjective effects. Therefore, in this review, to avoid the pitfalls of these alternative explanations, we will refer to alterations in operant drug IVSA or ethanol consumption as changes in reinforcement. The operant self-administration paradigm is also amenable to studying the phenomenon of relapse. The most widely used animal model of relapse is the reinstatement paradigm [3]. While this model by no means perfectly mirrors the phenomenon of relapse in humans, it is considered to be the best model developed thus far [4]. In the reinstatement paradigm, following the achievement of stable levels of self-administration, animals undergo extinction training procedures, where the behavior that previously resulted in the delivery of the drug solution (i.e., lever press or nose-poke) no longer produces any consequences. As a result of this imposed drug unavailability (i.e., forced abstinence ), animals will gradually extinguish (i.e., reduce) the behavior that previously resulted in drug delivery. Once predesignated extinction criteria have been obtained (for example, presses on the active drug-delivering lever are reduced to less than 20% of those observed when the drug was available), it is possible to reinstate operant responding by presenting one of the three main types of stimuli that are known to evoke relapse in humans: exposure to stressors, presentation of drug-associated environmental cues, or brief exposure to the drug itself. Upon presentation of one or more of these stimuli, animals

3 Gass and Foster Olive Page 3 will resume performing the operant task that previously led to drug delivery. This resumption, or reinstatement, of performing of the operant task is commonly interpreted as drug-seeking behavior. However, it should be noted that during reinstatement testing, the operant task does not result in actual drug delivery, so as to avoid the psychoactive effects produced by the drug which can confound interpretation of changes in drug-seeking behavior. This exemplifies one of the main divergences between the reinstatement model and relapse in humans, as the latter most often results in drug consumption. Another animal model of drug addiction is the conditioned place preference (CPP) paradigm [3]. Although it is widely used, this paradigm does not measure active drug seeking or reinforcement; rather, it utilizes Pavlovian conditioning procedures to provide an index of the rewarding (or subjective pleasurable) effects of drugs of abuse based on the animal s preference for a drug-paired environment over a non-drug paired environment. A typical CPP apparatus consists of two conditioning compartments with unique sensory characteristics (i.e., visually distinct wall patterns, flooring with unique tactile properties, or distinct olfactory cues). These two conditioning compartments are often connected by a neutral center start compartment. In a typical CPP experiment, an animal undergoes baseline preference testing and habituation, where it is placed in the center start compartment and allowed free access to both conditioning chambers for a specified amount of time. This allows for the animal to habituate to the testing environment as well as for the experimenter to determine if the animal exhibits any innate bias towards one of the two conditioning compartments (an ideal CPP apparatus would produce no innate preferences for either compartment). Then, the animal is subject to conditioning proceures where the conditioning drug (i.e., morphine, cocaine, amphetamine, etc.) is administered and the animal is confined to one of the two conditioning compartments for a specific amount of time. On alternate days, the animal is injected with saline or vehicle and then confined to the other conditioning compartment for the same amount of time. These conditioning sessions are repeated in an alternating fashion over a period of several days to allow the animal to form associations between the unique physical characteristics of the drug-paired compartment and the subjective effects of the conditioning drug. Finally, on the test day, the animal is placed back in the center compartment and allowed free access to both conditioning compartments. If the animal spends significantly more time in the drug-paired compartment compared to the saline-paired compartment, a CPP has been acquired. A conditioned place aversion (CPA) is observed if the animal spends significantly less time in the drug-paired compartment as compared with the saline-paired compartment. Drugs with aversive subjective properties, such as lithium chloride, or withdrawal from chronic drug administration reliably produce CPA. The CPP paradigm has provided substantial information on the neural substrates of the rewarding effects of drugs of abuse. One advantage of this paradigm is that the procedures are relatively simple, inexpensive, and less time-consuming to conduct than intravenous drug selfadministration. In addition, CPP paradigms can also be used to model various aspects of relapse. This is accomplished by extinguishing an established CPP by repeatedly pairing the previous drug-paired compartment with saline, or by allowing the CPP to dissipate over time with repeated testing of place preference. Then, pharmacological or other experimental manipulations can be introduced that result in a reinstatement of the original CPP. A disadvantage of the CPP technique, however, is that it does not directly measure drug-seeking behavior, but rather the motivation for secondary reinforcers (i.e., drug-associated environments) [3]. In addition, attempts to manipulate drug CPP via pharmacological or genetic methods do not always predict effects of those manipulations on drug self-administration behavior [3,5,6].

4 Gass and Foster Olive Page 4 3. Glutamatergic neurotransmission Glutamate is the most abundant excitatory neurotransmitter in the brain, mediating as much as 70% of synaptic transmission within the central nervous system and reaching extracellular concentrations in the low millimolar range. A diagram of a typical glutamatergic synapse is shown in Figure 2. Glutamate is packaged into synaptic vesicles in the presynaptic terminal by vesicular glutamate transporters (vgluts) using a proton gradient generated by the hydrolysis of adenosine triphosphate (ATP). Thus far, three different vgluts have been identified (vglut1-3) [7]. Once released into the synaptic cleft, glutamate can bind to one of three different types of ionotropic glutamate receptors (iglurs) located on the head of the postsynaptic spine: the N-methyl-D-aspartate (NMDA) receptor, the α-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) receptor, and the kainic acid (kainate, KA receptor). iglurs are ligand-gated ion channels that mediate fast excitatory neurotransmission. Glutamate can also bind to metabotropic glutamate receptors (mglurs) located in perisynaptic regions or on the presynaptic terminal (see Figure 2). NMDA receptors are heterotetrameric protein complexes that form ligand-gated ion channels composed of at least one NR1 subunit (for which there are at least 8 splice variants) and a combination of NR2A-D and NR3A or 3B subunits [8-10] (In mice, the NR1 subunit was previously been named ζ1 and the NR2A-D were previously named ε1-4). In addition to being stimulated by glutamate, amino acids such as D-serine and glycine act as co-agonists at the NMDA receptor. The NR2 subunits contain the glutamate binding domain, whereas the NR1 subunit contains the glycine-binding dopamine. Under resting conditions, the NMDA receptor channel pore is blocked by Mg 2+ ions, but once sufficient membrane depolarization has been established (i.e., by opening of AMPA receptor channels), the Mg 2+ block is removed, allowing the influx of cations (primarily Ca 2+ ions, but the NMDA receptor is also permeable to K + and Na + ions). Activity of the NMDA receptor is modulated by polyamines and inhibited by Zn 2+. The subunit composition of NMDA receptors are ontogenetically regulated and are neuroanatomically distinct. Once thought to be exclusively located on neurons, NMDA receptors have recently been shown to be expressed on glial cells including microglia, astrocytes and oligodendrocytes [10]. NMDA receptor subunits have also been found to exist on presynaptic terminals [11]. The NMDA receptor has been extensively implicated in mediating neural plasticity as well as learning and memory processes [12-14]. AMPA receptors are also heterotetrameric protein complexes that form ligand-gated ion channels composed of various subunits termed GluR1-4 (also termed GluRA-D) and GluRδ1 and 2 [8]. The mrnas encoding AMPA subunits can be edited or alternatively spliced to form variants such as the flip and flop isoforms. Each GluR subunit contains a binding site for glutamate. Once activated, AMPA receptors are permeable to various cations including Ca 2+, Na + and K +, although the majority of AMPA receptors in the brain contain GluR2 subunits, which render the channel impermeable to Ca 2+. Similar to NMDA receptors, AMPA receptor function can be modulated in the presence of polyamines. It is believed that both NMDA and AMPA receptors are necessary for the induction of many forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) [15-21]. Like NMDA and AMPA receptors, kainic acid (kainate, KA) receptors are also tetrameric protein complexes that form ligand-gated ion channels composed of various subunits. These subunits are termed GluR5-7 and KA1 and 2 [8]. KA receptors can form homomeric tetramers composed entirely of GluR5, 6 or 7 subunits or heteromeric complexes containg GluR5 or KA subunits. KA receptors are permeable to Na + and K + ions and, like NMDA and AMPA receptors, contribute to excitatory postsynaptic currents. The role of KA receptors in synaptic plasticity is less well-defined, however, but KA receptors have been found to be localized presynaptically where they can modulate neurotransmitter release [22].

5 Gass and Foster Olive Page 5 In addition to the iglurs, glutamate can also bind to mglurs, which are located either in the perisynaptic annulus or on presynaptic terminals. mglurs are seven transmembrane domain G-protein coupled receptors (GPCRs) that mediate slower, modulatory glutamatergic transmission. mglurs can be divided into three distinct groups, based on their pharmacological and signal transduction properties. Group I mglur receptors (mglur1 and mglur5) activate the Gα q class of G-proteins, which stimulate one of several phospholipases (including phospholipase C), resulting in phosphoinositol hydrolysis and the formation of lipid signaling intermediates such as inositol triphosphate (IP 3 ) and diacylglycerol (DAG), which in turn can activate various intracellular messengers including protein kinase C (PKC) [23-25]. Activation of Group I mglur receptors also mobilizes calcium release from IP 3 receptor-mediated intracellular stores, which can in turn activate other intracellular messengers such as calmodulin-dependent kinase II (CaMKII). Group I mglurs, particularly mglur5, are positively coupled to NMDA receptor function via PKC, and are structurally linked to these receptors as well as IP 3 -gated intracellular Ca 2+ stores via the Homer family of proteins [26-30]. Group I mglurs are rarely found presynaptically. Group II (mglur2 and mglur3) and Group III (mglur4, mglur6, mglur7, and mglur8) mglurs, on the other hand, activate the Gα i class of G-proteins and are negatively coupled to adenylyl cyclase (AC) activity, and upon stimulation result in decreased intracellular levels of cyclic adenosine monophosphate (camp). Presynaptically localized Group II and Group III mglurs, particularly mglur2 and mglur3, are thought to represent the classical inhibitory autoreceptor mechanism that suppresses excess glutamate release. mglur3 and mglur5 have been localized to glial cells such as astrocytes [31]. Activation of iglurs alone is sufficient for the propagation of the action potential by the postsynaptic neuron, and can activate various intracellular signaling molecules including protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and extracellular signalrelated kinase (ERK) [32] (see Fig. 2). Activation of additional signaling molecules, such as PKC, is achieved by activation of mglurs. Together, the simultanous activation of iglurs and mglurs activates a host of intracellular signaling pathways that result in protein phosphorylation of ion channels, other kinases, and transcription factors and eventually leads to the molecular events underlying neural plasticity. Such events include initiation and/or regulation of dendritic mrna translation and de novo protein synthesis, changes in gene expression in the nucleus, and cytoskeletal remodeling (Fig. 2) [33]. Glutamate-mediated neural plasticity is also characterized by changes in iglur subunit trafficking such as insertion of AMPA receptors into postsynaptic plasma membrane (see Section 11). Glutamate is cleared from the extracellular environment by a family of sodium-dependent excitatory amino acid transporters (EAATs). To date, five separate EAATs have been idenfitied (EAAT1-5). EAAT1-3 are alternatively termed GLAST, GLT-1, and EAAC1, respectively. EAAT2 and EAAT5 are localized to the presynaptic terminal, EAAT3 and EAAT4 are localized to the postsynaptic neuron, and EAAT1 and EAAT2 are expressed in glial cells [7]. This family of EAATs provides numerous mechanisms to prevent an excessive accumulation of extracellular glutamate, which if high enough concentrations are reached, can result in excitotoxicity. Once inside glial cells, glutamate is converted to glutamate by glutamine synthetase, where it is secreted back outside the glia and taken up by the presynaptic terminal for conversion back to glutamate by glutaminase (Fig. 2). Conversely, glutamate can be transported from within glial cells to the extracellular environment by the cystine-glutamate exchanger (x c ) [34-37]. As mentioned earlier, glutamatergic transmission accounts for up to 70% of synaptic transmission in the central nervous system. Thus, there are glutamatergic projections and/or neurons expressing glutamate receptors in numerous circuitries of the brain, including the mesolimbic dopamine reward circuitry. This reward circuitry is composed primarily of

6 Gass and Foster Olive Page 6 dopamine-synthesizing neurons in the VTA of the ventral midbrain that project rostrally to target regions such as the nucleus accumbens (NAcc), amygdaloid complex (Amyg) and frontal cortex (FC). Each of these regions receive substantial glutamatergic input [38,39] (Fig. 3). For example, the VTA receives glutamatergic projections from the FC, Amyg, pendunculopontine tegmentum (PPT), and laterodorsal tegmentum (LDT) [40,41]. A recent report indicated that a subpopulation of VTA neurons express vglut2 mrna [42], suggesting the existence of neurons intrinsic to this region that utilize glutamate as a neurotransmitter. The NAcc receives a convergence of glutamatergic input from the FC, Amyg, hippocampal formation (Hipp), and various nuclei of the thalamic (Thal). The FC cortex receives glutamatergic input from the Hipp, Amyg and Thal. Thus, there is a robust excitatory glutamatergic innervation of the mesolimbic dopamine reward circuitry which proves and anatomical basis for dopamineglutamate interactions in regulating the addictive properties of drugs of abuse as well as synaptic plasticity [43-45] (see Section 11 for further discussion of this topic). In light of all the receptor proteins and transporters associated with glutamatergic neurotransmission, a substantial array of pharmacological ligands has become available to researchers attempting to examine the role of glutamatergic transmission in preclinical models of drug addiction. Table 1 details some of the more commonly used glutamatergic ligands that are employed in preclinical addiction research. 4. Glutamate and cocaine Cocaine is a potent inhibitor of presynaptic monoamine transporter function, and as a result produces substantial increases in extracellular levels of dopamine, serotonin and norepinephrine in the synaptic cleft, particularly in forebrain terminal fields that receive monoamineric projections. Cocaine also acts as a local anesthetic by blocking sodium channels and thereby inhibiting the propagation of action potentials. Some of the first observations that cocaine interacts with glutamatergic systems in the brain were reported in the late 1980 s and early 1990 s, when several groups of investigators showed that that sensitization to the locomotor stimulant effects of cocaine was blocked by pretreatment of the NMDA antagonist MK-801 [46], and that infusion of iglur antagonists into the NAcc reduced the motor stimulant and reinforcing effects of cocaine [47-51]. Since these observations, a tremendous amount of evidence has accumulated indicating an important role for glutamate in the motor stimulant properties of cocaine and its role in locomotor sensitization, and the reviewer is directed to various reviews elsewhere for in depth details on this topic [52-60]. Here we will focus on the effects of cocaine on glutamatergic transmission as well as the effects of glutamatergic ligands on the rewarding and reinforcing effects of cocaine. In addition to elevating extracellular levels of monoamines, in vitro release or in vivo microdialysis studies have shown that cocaine also increases extracellular levels of glutamate in various brain regions including the dorsal striatum [61], NAcc [62-71], septum [72], ventral pallidum [73,74], VTA [75,76] and cerebellum [77]. However, it should be noted that not all studies have observed cocaine-induced increases in extracellular glutamate, including studies in self-administering animals [78]. Others have found stimulatory effects of cocaine on extracellular glutamate only with high (i.e., 30 mg/kg i.p.) doses of cocaine [63,65,66], which may cause neurotoxicity. Thus, the ability of cocaine to induce increases in extracellular glutamate is not a universally observed phenomenon. Nonetheless, immunohistochemical studies have shown that cocaine decreases glutamate immunoreactivity in presynaptic terminals several brain regions including as the NAcc [79-81], suggesting that some of the observed elevations in extracellular glutamate in response to cocaine are derived from neuronal stores. Several reports suggest that environmental contexts and cues are important modulatory factors in the ability of cocaine to elevate extracellular levels of glutamate in the NAcc [67, 82] but not the frontal cortex [83].

7 Gass and Foster Olive Page 7 Glutamate mediates numerous neuronal effects of acute exposure to cocaine. Low doses of acutely administered cocaine enhance glutamate-evoked neuronal firing in the cortex, striatum, or NAcc, whereas higher or self-administered doses tend to inhibit glutamate-evoked neuronal activity in these regions [84-86]. However, the ability of high concentrations of locally administered cocaine to suppress glutamate-induced neuronal activity should be interpreted with caution, since cocaine exerts local anesthetic effects via blockade of voltage-gated sodium channels. Drugs of abuse including cocaine can produce elevated resting neuronal membrane potentials (i.e., up states) in regions such as the dorsal striatum, NAcc, and FC, and this phenomenon requires coordinated interactions between dopaminergic and glutamatergic transmission [87,88]. The ability of acute cocaine exposure to increase striatal neuropeptide expression and activate signaling molecules such as ERK is dependent on NMDA receptor activation [89-91]. Acute administration of cocaine has been shown to increase phosphorylation of the GluR1 subunit of the AMPA receptor in the striatum [92], induce a redistribution of AMPA and NMDA receptors in the VTA [93-95], and induce the formation of NR2B-D2 heteroreceptor complexes [96]. The expression of various iglur subunits is reduced by acute cocaine. For example, in situ hybridization studies have shown that mrna levels for GluR3, GluR4 and NR1 are decreased in the NAcc by acute cocaine exposure, as is NR1 mrna expression in the striatum and VTA [97], perhaps as a compensatory response to increased glutamate overflow produced by cocaine. However, acute cocaine has been shown to increase NR1 mrna expression in the hippocampus [98] and GluR2 mrna in the striatum [99]. Repeated cocaine exposure can lead to a phenomenon called behavioral sensitization (sometimes termed reverse tolerance ), which is a progressive increase in the behavioral (i.e., locomotor) response to cocaine in response to repeated exposure to the same dose. Behavioral sensitization to cocaine is paralleled by adaptive changes in mesolimbic dopamine system function as well as the responsiveness of this system to glutamate. For example, repeated exposure of rats to cocaine or amphetamine results in a transient enhanced responsiveness of VTA dopamine neurons to locally applied glutamate [100], and it was later determined that this phenomenon was a result of increased responsiveness of AMPA and not NMDA or mglur receptors located on VTA dopamine neurons [101]. Such an effect is paralleled by an increase in expression of GluR1 receptor levels in the VTA, but not the substantia nigra, following repeated cocaine exposure [ ]. Expression of NR1 in the VTA has also been reported to be increased following repeated cocaine, but the expression of NR2A/B, GluR2/3 or GluR6/7 is unaltered [ ]. Some investigators have not observed changes in iglur expression in the VTA [97, ]. These discrepancies may be due to the type of detection methodology used (i.e., immunoblotting, radioimmunohistochemistry, or in situ hybridization), variations in cocaine dose, length of withdrawal period prior to analysis, or the inclusion of a final challenge administration of cocaine in order to demonstrate the maintenance of behavioral sensitization. Nonetheless, post-mortem analysis of human brain tissue samples from cocaine overdose victims did reveal an upregulation of numerous iglur subunits in the VTA [109,110], suggesting that perhaps cocaine, at least at high doses, alters iglur subunit expression in the VTA. In the primary target field of VTA DA neurons, the NAcc, it has been demonstrated that multiple cocaine exposures result in a sensitized increase in extracellular levels of glutamate [64,65] but see [111], paralleled by an accumulation of presynaptic glutamate immunoreactivity in this region [112]. Changes in glutamate receptor expression in the NAcc following repeated cocaine exposure are, however, complex and inconsistent across studies. Some investigators have reported no changes in expression of NR1, NR2A/B, GluR1-2, or KA receptor subunits in the NAcc within 24 hr of discontinuation of cocaine treatment [102,103], or have reported decreases in NR1 and GluR3/4 expression [97,98]. However at later time

8 Gass and Foster Olive Page 8 points, others have observed increases in NR1 expression in this region [97,103,113], but only in rats that exhibited signs of behavioral sensitization. Reductions in NR2B expression in the NAcc were observed after 24 hr [114] but not 1 week [115] of cocaine withdrawal. In an elegant protein cross-linking study, it was demonstrated that repeated cocaine exposure increases the suface expression of GluR1-3 subunits in the NAcc at 3 weeks following discontinuation of treatment, but only in rats showing behavioral signs of sensitization [116]. Collectively, these data show that the effects of repeated cocaine exposure on iglur subunit expression can vary considerably depending on many experimental factors, and that any lack of observed changes in overall protein expression may overlook more subtle adaptive changes such as increased surface expression of AMPA receptor subunits. All of the aforementioned studies on the effects of repeated cocaine administration on iglur expression utilized response non-contingent (i.e., passive, experimenter-administered) administration of the drug. Interestingly, a recent study by Hemby and colleagues examined the effect of active intravenous cocaine self-administration on changes in iglur subunit expression in the NAcc [108]. It was found that during the early phases of cocaine withdrawal, NR1 and GluR5 expression was reduced in the VTA, no changes in any subunit examined were observed in the NAcc, and in the frontal cortex NR1 levels were increased and GluR2-6 and KA2 levels were decreased. Although it is difficult to compare these results with those reviewed above due to the many procedural differences involved, this study was one of the first to examine changes in iglur subunit expression in addiction-related brain regions following active self-administration of the drug. During protracted cocaine withdrawal, extracellular levels of glutamate and presynaptic glutamate immunoreactivity are decreased in the NAcc [36,37,64,67,69,79-81] but increased in the prefrontal cortex [117]. This reduction in extracellular levels of glutamate in the NAcc may be due to desensitization of presynaptic Group II mglurs [ ] as well as a downregulation of x c function [36], which tightly regulate extracellular glutamate levels. As a result, increases in NR1, GluR1-3, and Group I mglur expression have been observed in the NAcc [97,103,104,122,123], suggesting that adaptations to reduced extracellular glutamate levels may occur. The observed decreases in extracellular glutamate during cocaine withdrawal have been shown to be critically involved in relapse-like behavior, since pharmacological restoration of basal extracellular glutamate levels to those observed in non-withdrawn animals, as with agents such as N-acetylcysteine that promote the activity of x c (see Fig. 4 and Section 13.2), inhibit the ability of cocaine priming to further increase extracellular levels glutamate levels in the NAcc. The ability of N-acetylcysteine to inhibit cocaine-induced increases in NAcc glutamate overflow is paralleled by blockade of cocaine-induced reinstatement [37,69], suggesting that an imbalance in glutamatergic transmission in the NAcc is may induce a predisposition towards relapse. Other changes brought on by repeated cocaine exposure include: an upregulation of NMDA receptor binding in the regions such as the cortex, striatum, amygdala and hippocampus has been reported following repeated cocaine exposure [ ] but see [127] a decrease in NR1 and/or NR2B/2C expression in regions such as globus pallidus, subiculum, striatum, and cerebellum [98,105] an up-regulation of NR1 and a down-regulation of GluR2-7 and KA2 expression in cerebral cortex [97,108,128] increased phosphorylation of GluR1 in the prefrontal cortex [115] an increase in mglur5 expression in the hippocampus [129]

9 Gass and Foster Olive Page 9 bidirectional alterations in the expression of NMDA and AMPA receptor subunit expression in the amygdala [130,131] A role for glutamatergic transmission in the rewarding and reinforcing effects of cocaine has been clearly demonstrated by pharmacological studies utilizing iglur antagonists. Systemic administration of NMDA antagonists attenuate cocaine reinforcement [ ] and the acquisition and/or expression of a cocaine CPP [136,137]. Similar reductive effects on cocaine reinforcement have been reported following administration of inhibitors of glutamate carboxypeptidase II [138], which reduce free glutamate availability. The ability of iglur antagonists to reduce cocaine reinforcement are likely mediated, at least in part, by NMDA and/or AMPA receptors in the NAcc and dorsal striatum, as evidenced by microinjection studies [49,139,140] but see [141]. Stimulation of AMPA receptors in the NAcc has been shown to reinstate previously extinguished cocaine-seeking behavior [ ], and AMPA antagonists infused into the NAcc block reinstatement induced by priming injections of cocaine [142], cocaine-associated cues [144], or infusions of cocaine into the FC [145]. On the other hand, some studies have reported that NMDA receptor antagonists, whether administered systemically or into the NAcc shell, actually induce reinstatement of cocaine-seeking behavior [ ]. The reason for the opposing effects of NMDA and AMPA antagonists on reinstatement are currently unclear, but may involve cocaine-induced increased AMPA receptor responsiveness (relative to NMDA) due to trafficking of AMPA subunits to the plasma membrane [116]. The FC has been identified as the primary source of glutamatergic afferents to the NAcc that mediate cocaine-primed reinstatement [68]. Together these data suggest a critical role for iglurs in the NAcc in mediating the reinforcing effects of cocaine as well as the ability of exposure to cocaine or drug-associated cues to reinstate cocaine-seeking behavior (see also [148,149]). However, further research into identifying the unique, and possibly opposing, contributions of AMPA and NMDA receptors in the NAcc to reinstatement of cocaine-seeking behavior is needed. Glutamatergic transmission in the VTA also plays a role in cocaine reward, reinforcement and reinstatement. For example, electrical stimulation of glutamatergic fibers in the ventral subiculum reinstates cocaine-seeking behavior in a manner dependent on glutamate transmission in the VTA [150]. Blockade of NMDA or AMPA receptors in the VTA blocks the development of cocaine CPP [151] and cue-induced reinstatement [152]. Expression and phosphorylation of GluR1 in the VTA also mediates the reinforcing effects of cocaine [153]. The basolateral amygdaloid nucleus (BLA) also plays a role in cue-induced reinstatement of cocaine-seeking behavior (reviewed in [154]). However, conflicting results have emerged as the the role of glutamate transmission in this phenomenon. For example, infusion of NMDA into the BLA reinstates previously extinguished cocaine-seeking behavior [155], yet infusions of iglur antagonists in this region apparently do not attenuate cue-induced reinstatement [156]. Thus, while glutamatergic transmission in this region may play a role in reinstatement per se, iglurs apparently do no mediate the ability of drug-associated cues to induce reinstatement. In addition, AMPA receptors in the NAcc mediate cocaine-seeking behavior only when BLA dopamine receptors are blocked [157] mglurs, particularly Group I and Group II mglurs, are also involved in cocaine reward and reinforcement. Following a pivotal study by Chiamulera and colleagues who showed that mice carrying a targeted deletion of the mglur5 gene do not self-administer cocaine [158], numerous investigators have shown that mglur5 antagonists reduce cocaine reinforcement and reinstatement [ ]. mglur5 receptor antagonists also reduce the development and/ or expression of cocaine CPP [166,167]. Dampening glutamate transmission via stimulation of presynaptic mglur2/3 receptors or activating glutamate transporters attenuates cocaine reinforcement [168,169], cue- and cocaine-induced reinstatement [168,170], incubation of

10 Gass and Foster Olive Page 10 cocaine craving (i.e., a progressive increase in the magnitude of cue-induced reinstatement over time following cocaine self-administration) [171], and the development of cocaine CPP [172]. The NAcc and amygdala appear to be important mediators of some of these effects [144,169,171]. In addition to the aforementioned pharmacological evidence, studies utilizing genetically modified mice have also shed light on the role of glutamatergic transmission in cocaine addiction, as summarized below: genetic deletion of GluR1 and/or GluR2 does not alter the acquisition of cocaine CPP [173,174], but impairs extinction of cocaine-seeking behavior [174]; however, another group of investigators showed a failure of GluR1 null mutant mice to develop cocaine CPP [175] cocaine CPP is attenuated in mice engineered to express an NR1 subunit with reduced cation flux properties explicitly in D 1 -containing neurons [176] Mice carrying a targeted deletion of the mglur5 gene do not self-administer cocaine and are indifferent to its locomotor stimulant effects [158] genetic deletion of mglur2 enhances cocaine CPP [71]. Altered behavioral and neurochemical responses to cocaine are observed in Homer1 or Homer2 knockout mice, as described below. Homer proteins are a family of scaffolding proteins that link NMDA, Group I mglurs and IP 3 -gated intracellular calcium stores to the postsynaptic density. A growing body of literature suggests that the expression of these proteins is regulated by cocaine and that they play an important role in the behavioral and neurochemical effects of this psychostimulant. Acute cocaine treatment induces a rapid but transient increase in Homer 1a but not Homer1b/c or Homer 2a/b expression in the dorsal striatum [177]. Chronic cocaine treatment, on the other hand, reduces Homer 1b/c levels in the NAcc [119]. In addition, antisense knockdown of Homer1 expression in this region sensitizes rats to the locomotor stimulant effects of cocaine and also downregulates GluR1 expression [178], suggesting that cocaine-induced downregulation of Homer1 proteins may regulate its behavioral sensitizing effects. Subsequently, it was shown that mice carrying a targeted deletion of either the Homer1 or Homer2 gene demonstrate increased locomotor stimulant and rewarding effects (as measured by CPP) of cocaine as compared to wildtype controls [ ], thus exhibiting a behavioral phenotype similar to that of cocaine-sensitized animals. In addition, Homer2 deficient mice exhibited a more rapid acquisition of cocaine IVSA and decreased basal extracellular glutamate levels in the NAcc (similar to cocaine withdrawn animals). Remarkably, many of these behavioral and neurochemical changes could be reversed by virally-mediated restoration of Homer2 in the NAcc [180]. These findings highlight the importance of Homer proteins and the functional consequences of alterations in their expression as a result of repeated cocaine exposure. 5. Glutamate and amphetamines Amphetamines are psychomotor stimulant drugs that promote the release of monoamines by reversing the directionality of vesicular and plasmalemmal monoamine transporters located in presynaptic terminals, thereby promoting the release of dopamine, norepinephrine and serotonin into the synaptic cleft. Although there are many amphetamine-related compounds, the primary analogues that will be focused on in this review are d-amphetamine (herein referred to as amphetamine ), methamphetamine, and methylenedioxymethamphetamine (MDMA, Ecstasy ).

11 Gass and Foster Olive Page 11 As with cocaine, the first observations that glutamate was involved in the psychomotor stimulant properties of amphetamine were reported in the late 1980 s, when it was demonstrated that sensitization to the locomotor stimulant effects of amphetamine was blocked by co-administration of the NMDA antagonist MK-801 [46], and that infusion of iglur antagonists into the NAcc reduced the motor stimulant effects of amphetamine [47]. The reader is directed to various reviews elsewhere for in depth details on the role of glutamate in amphetamine-induced locomotor activity and behavioral sensitization [52-60]. In addition, the reader is also directed to several recent reviews on the role of glutamate in amphetamineinduced neurotoxicity [ ]. In addition to being monoamine-releasing agents, amphetamines also elevate extracellular levels of glutamate, as demonstrated by in vitro release and in vivo microdialysis studies in the cerebral cortex [66, ], dorsal striatum [187,191] [ ] but see [203], NAcc [66, 190, ], hippocampus [187,208,209] and VTA [205,210,211]. However, it should be noted that some of these studies used doses of amphetamine that were in the neurotoxic range (i.e., 9-10 mg/kg i.p.) [193,204]. On the other hand, high doses of methamphetamine are often intentionally utilized to explore the hypothesis that prolonged increases in extracellular glutamate levels underlie the neurotoxic effects of this drug (c.f., [192,195,196,201,202, 212]). Presynaptic glutamate immunoreactivity has been shown to be decreased in various brain regions following methamphetamine administration [213,214], suggested that some of the elevations in extracellular glutamate induced by this drug are derived from neuronal stores. Elevation of extracellular glutamate by amphetamines in various brain regions is not likely a result of alterations in EAAT function [215,216], although one study implicated EAATs in the ability of amphetamine to increase glutamate overflow in the VTA [211]. Some investigators have shown that local administration of high concentration of amphetamines actually reduce extracellular glutamate levels in certain brain regions [193,217,218] and suppress glutamate evoked neuronal activity in the NAcc and cerebral cortex [219,220]. Acute administration of amphetamines has been consistently shown to increase the expression immediate early genes such as c-fos and Zif268, various neuropeptide precursors, and induce phosphorylation of various transcription factors in the dorsal striatum, all of which are dependent on iglur- and/or mglur-mediated mechanisms [89, ]. Oddly, however, amphetamine-induced increases in the expression of immediate early genes in the NAcc appear to be NMDA-receptor independent [230]. Acute administration of methamphetamine has been shown to increase phosphorylation of the GluR1 subunit of the AMPA receptor in the striatum [92], which alters AMPA receptor channel conductance and promotes trafficking to the plasma membrane. With regards to VTA dopaminergic neurons, acute administration of amphetamines generally tends to inhibit the firing of these neurons by inducing somatodendritic release of dopamine, which in turn stimulates inhibitory D 2 -like autoreceptors. However, it has also been shown that acute amphetamine may actually excite VTA dopamine neurons by inhibition of mglur-mediated inhibitory postsynaptic currents [231], Amphetamine can also induce cellular hallmarks of neural plasticity in these neurons, such as increasing the AMPA component of evoked excitatory postsynaptic currents [ ] Repeated exposure of amphetamines can have markedly different effects on glutamatemediated neuronal activity and function as compared with acute exposure. For example, as mentioned above, acute administration of amphetamine can suppress glutamate evoked neuronal activity in the NAcc and cerebral cortex [219,220]. However, repeated amphetamine administration results in enhanced neuronal responsiveness to locally applied glutamate in the VTA [100,101] and frontal cortex [235] but not NAcc [100]. It is unclear if the mechanisms underlying the enhanced responsiveness to glutamate are the same across these regions.

12 Gass and Foster Olive Page 12 Repeated amphetamine exposure also results in various changes in other components of glutamate transmission. For example, repeated methamphetamine administration produces a reduction in NMDA NR1, NR2A and NR2B protein levels in the striatum [236] but increases vglut1 levels in this region, which may facilitate the incorporation of glutamate into synaptic vesicles to promote long-lasting methamphetamine-induced increases in extracellular glutamate [202]. Repeated exposure to amphetamine also produces changes in AMPA receptor expression which can be either short- or long-lasting. In a series of studies conducted by Wolf and colleagues, it was shown that 5 days of amphetamine treatment produced decreases in mrna and protein levels of GluR1, GluR2 and NR1 in the NAcc and increased NR1 expression in the frontal cortex at 14 but not 3 days following that last amphetamine exposure [ ]. However, changes in GluR1 in the frontal cortex were only transient, being increased at 3 but not 14 days following the last amphetamine administration [237,238]. With regards to the VTA, it has been shown that repeated amphetamine treatment does not alter AMPA subunit expression [106,240], but does sensitize the ability of intra-vta applied AMPA to increase in extracellular levels of glutamate in this region [241], suggesting a sensitizing effect of repeated amphetamine on AMPA receptor function in the VTA without alteration in subunit expression. However, many of these studies examined receptor protein levels in tissue homogenates by immunoblotting, or immunoreactivity or mrna expression at the level of the cell body. Thus, it is possible that more subtle changes in receptor levels, such as increased surface expression of AMPA receptor subunits as has been observed following cocaine treatment [116], may be overlooked by use of these techniques. Repeated amphetamine also alters the expression of mglurs, causing transient increases mglur1 expression in the dorsal and ventral striatum, but more persistent reductions in mglur5 expression in these regions [242]. Repeated amphetamine also increases hippocampal and cortical mglur5 expression [243,244]. Repeated amphetamine administration does not alter EAAT2 or EAAT3 expression in various regions including the midbrain, NAcc, dorsal striatum or FC [245,246]; however, methamphetamine has been shown to increase the expression of EAAT2 in the striatum [247]. Glutamate appears to play an important role in the rewarding and reinforcing effects of amphetamines. Reductions in glutamate transmission by administration of the glutamate release inhibitor riluzole [248], the glutamate transporter activator MS-153 [172], infusion of an AMPA/KA antagonist into the NAcc [249], and virally-mediated overexpression of EAAT2 in the NAcc [250] all have been shown to attenuate the development of amphetamine CPP. However, it was also found that antagonism of mglur2/3 receptors, which facilitates glutamate transmission, disrupts the ability of intra-nacc amphetamine to establish a CPP [251], suggesting that excessive glutamatergic transmission may actually disrupt the neuronal communication that normally subserves the ability of amphetamine to establish a CPP. Our laboratory demonstrated that the development of amphetamine CPP was not attenuated by the mglur5 antagonist MPEP [166]. However, others have shown that the expression of amphetamine CPP, but not MDMA CPP, is suppressed by MPEP [252]. With regards to amphetamine reinforcement and relapse-like behavior, surprisingly little attention has been given to the potential role of glutamate. Both dextromethorphan and the African tree shrub extract ibogaine, which have NMDA receptor antagonist properties, reduce methamphetamine reinforcement [253,254] and the establishment of amphetamine CPP [255]. However, ibogaine has been reported to have numerous other neurochemical effects including nicotinic acetylcholine receptor (nachr) antagonism [256], and current evidence from studies with ibogaine congeners that are devoid of NMDA antagonist activity indicate that the inhibitory effects of ibogaine on amphetamine reward and reinforcement are likely mediated by antagonism of nachrs rather than NMDA receptor blockde [257]. However, one recent study did show that stimulation of mglur2/3 receptors attenuates enhanced